Landscape factors and allochthonous congeneric species influence Callithrix aurita occurrence in Brazilian Atlantic Forest remnants

Abstract The buffy‐tufted‐ear marmoset (Callithrix aurita) is a small primate endemic to the Brazilian Atlantic Forest biome, and one of the 25 most endangered primates in the world, due to fragmentation, loss of habitat, and invasion by allochthonous Callithrix species. Using occurrence data for C. aurita from published data papers, we employed model selection using Akaike Information Criterion corrected for small samples and cumulative AICc weight (w +) to evaluate whether fragment size, distance to fragments with allochthonous species, altitude, connectivity, and surrounding matrices influence the occurrence of C. aurita within its distributional range. Distance to fragments with C. jacchus (w + = 0.94) and nonvegetated areas (w + = 0.59) correlated negatively with C. aurita occurrence. Conversely, the percentage of agriculture and pasture mosaic (w + = 0.61) and the percentage of savanna formation (w + = 0.59) in the surrounding matrix correlated positively with C. aurita occurrence. The findings indicate that C. aurita is isolated in forest fragments surrounded by potentially inhospitable matrices, along with proximity of a more generalist and invasive species, thereby increasing the possibility of introgressive hybridization. The findings also highlighted the importance of landscape elements and allochthonous congeneric species for C. aurita conservation, besides indicating urgency for allochthonous species management. Finally, the approach used here can be applied to improve conservation studies of other endangered species, such as C. flaviceps, which is also endemic to the Brazilian Atlantic Forest and faces the same challenges.


| INTRODUC TI ON
The conversion of native habitats into anthropic landscapes, and the accompanying habitat fragmentation and loss, poses a challenge for primate conservation efforts (Estrada et al., 2017;Foley et al., 2005). These processes change environmental integrity (e.g., heterogeneity and structure previous to human activities), on a landscape scale and can increase isolation and reduce large extensions of habitat into smaller fragments, which are immersed in altered matrices (Andrén, 1994;Hanski, 2015). The effects of habitat loss on species are potentiated when they are associated with human activities, such as logging, agriculture, hunting, and trafficking (Estrada et al., 2017). Anthropogenic activities that promote fragmentation can also isolate protected areas and increase susceptibility to invasion by allochthonous species (Spear et al., 2013), which represent another major threat to species conservation (Butchart et al., 2010).
Neotropical primates constitute an excellent model to evaluate the effects of landscape changes since these animals are arboreal and, in general, ecologically less flexible than terrestrial mammals in terms of vegetation cover loss (Arroyo-Rodríguez & Mandujano, 2009;Isaac & Cowlishaw, 2004). Studies show that primate ecology such as distribution, diet, home range, and even social organization can be affected at several levels (Arroyo-Rodríguez et al., 2008;Cristóbal-Azkarate & Arroyo-Rodríguez, 2007;Zunino et al., 2007). Although some generalist primate species can perform landscape supplementation (Dunning et al., 1992), which means they can search and acquire resources in neighboring fragments or in the matrix itself (Asensio et al., 2009), landscape attributes directly influence the establishment of their populations in forest fragments (Arroyo-Rodríguez et al., 2008).
Soon after changes occur in forest cover, such as selective logging, primates tend to be randomly distributed in the landscape forest fragments. However, throughout the process, they end up colonizing only fragments that meet their environmental requirements (Chapman et al., 2003;Marsh, 2003). Such requirements may include a minimum fragment size according to their home range, and food resources. Some of these requirements are well-known for a few primate species (Arroyo-Rodríguez & Mandujano, 2009), but are relatively unknown for other species. Determining these requirements, and the consequent suitability of habitats, is important to understand the occupation (Arroyo-Rodríguez et al., 2008) and occurrence (Hoffman & O'Riain, 2012;Silva et al., 2015) of primate populations and, therefore, the development of plans for their conservation (e.g., Andrew & Ustin, 2010). Understanding such factors is especially important for species that inhabit areas with high levels of fragmentation (Robbins & McNeilage, 2003), as is the case of Callithrix species in the Atlantic Forest.
The conservation status of the genus Callithrix is worsened by hybridization between species, which generates fertile offspring with intermediate characteristics (Malukiewicz et al., 2015). This is one of the greatest current threats to the conservation of buffy-tufted-ear marmoset (Callithrix aurita) (Carvalho et al., 2018), a marmoset endemic to the Brazilian Atlantic Forest in the states of Minas Gerais, Rio de Janeiro, and São Paulo. As a biome characterized by high species diversity and a high degree of endemism, the Atlantic Forest is one of the main biodiversity hotspots in the world, despite being highly fragmented with only 12.4% of the original extension remaining (SOS Mata Atlântica, 2019). Habitat loss and fragmentation, allied with the co-occurrence of congeneric invasive species, are the main extinction threats for C. aurita (Malukiewicz et al., 2021). The International Union for Conservation of Nature (IUCN) Red List categorizes C. aurita as Endangered (Melo et al., 2021). Furthermore, the species is among the 25 most endangered primates of the world (Schwitzer et al., 2019) and is included in the National Action Plan for the Conservation of Atlantic Forest Primates and the Collared Sloth (PAN PPMA) (ICMBio/MMA, 2018). This species has more specialized characteristics regarding feeding behavior in comparison with C. penicillata and C. jacchus, such as having less capacity of treegouging to eat gum, which also restricts its distribution (Rylands et al., 2009). Its home range encompasses up to 35.3 ha (Corrêa et al., 2000), despite occurring in forest fragments with as low as 3 ha (Oliveira, 2012), and is generally found at altitudes above 500 m (Rylands & Faria, 1993).
The two most generalist species of the genus are C. jacchus and C. penicillata, the former originally ocurred in the Caatinga and in part of the Atlantic Forest in Northeast Brazil and the latter in the Cerrado (Hershkovitz, 1977;Raboy et al., 2008). However, the distributions of these species have expanded due to introduction processes in other regions of Brazil (Oliveira & Grelle, 2012;Rylands, 1993). They are extremely flexible ecologically, and so are categorized as Least Concern by the IUCN (Bezerra et al., 2018;Bicca-Marques et al., 2018). The use of gummivory (i.e., a diet based on plant exudates) with greater intensity makes them more generalist and facilitates their adaptation to new areas (Abreu et al., 2016;Vilela & Del-Claro, 2011). This factor also influences their home ranges, which reach only 0.5 ha for C. jacchus (Stevenson & Rylands, 1988) and 2.5 ha for C. penicillata (Fonseca & Lacher, 1984).
Few studies have shown landscape factors as predictors of the occurrence of Callithrix species (e.g., Flesher, 2015), especially on a regional scale. For C. aurita specifically, the literature is scarce (e.g., Silva et al., 2015). In this context, this work aimed to evaluate whether landscape elements, such as patch attributes and the occurrence of allochthonous congeneric species, influence the occurrence of C. aurita within its distribution. We hypothesized that the probability of occurrence of C. aurita would be: (1) positively influenced by forest fragment altitude, area, connectivity, and distance to allochthonous species (Arroyo-Rodríguez & Mandujano, 2009;Carvalho et al., 2018;Corrêa et al., 2000;Malukiewicz, 2019;Palacios, 2018;Rylands & Faria, 1993);

| Study area
The study was carried out within the original distribution of C. aurita, adapted based on the map provided by the IUCN (Melo et al., 2021),  Figure 1). The natural dispersion of these species to the study area is considered unlikely, considering the existence of barriers such as large discontinuous areas (Cerqueira et al., 1998;Morais et al., 2008).

| Occurrence data compilation
We used occurrence data from two data papers: "Atlanticprimates"  and "Neotropical alien mammals" (Rosa et al., 2020). Only occurrence records of the species of interest (C. aurita, C. jacchus, and C. penicillata) in the study area, and collected between 1985 and 2019, were included. This time window was used due to overlap with the land use and cover image database available in Atlantic Forest Collection 5 on the MapBiomas project platform (MapBiomas, 2020). These raster files are based on Landsat satellite images, with 30-m resolution. We excluded records collected in urban areas, in addition to those obtained by methods without empirical evidence (e.g., interviews). The remaining records were inserted into the QGIS software version 3.10.13 (QGIS, 2020) for checking the points and validation regarding overlap with the study area. In case of doubts about the geographic coordinates specified in the original worksheet, they were checked and, if necessary, corrected based on the coordinates explained in the referenced articles.
After filtering the data, 23 forest fragments containing records of occurrence of C. aurita were obtained and the same number of control fragments (i.e., without confirmed records of C. aurita) were randomly selected, for a total of 46 study fragments. Importantly, all control fragments were within the study area and belonged to Atlantic Forest areas.

| Landscape metrics
Landscape analysis within the study area employed QGIS software (QGIS, 2020), GRASS GIS version 7.8.4 (GRASS, 2020), and Fragstats Taking into account that (1) the spatial configuration of the Atlantic Forest biome did not change much in the last 30 years (the peak of changes in the biome was between 1950 and 1960; Fonseca, 1985); (2) we had, in some cases, registers of the species of interest for the same area in different years; and (3) the life expectancy of Callithrix species can be up to 21 years (Nishijima et al., 2012), we assumed that patches that were occupied when the data were collected for the first time would remain occupied in the subsequent years. Additionally, because we had a limited number of registers of the species of interest, we could not make separate analyses including specific time intervals. Thus, for the subsequent analysis, we combined all the registers of Callithrix species and averaged along the years the landscape metrics, named fragment area, Euclidean Nearest Neighbor Distance (ENN), the Proximity Index (PROX), and the surrounding matrix variables (see details below).
The average area of each study fragment was calculated on Fragstats software (Table 1), with input of raster images of land use and cover of the Atlantic Forest of the MapBiomas project (MapBiomas, 2020). We averaged area values from the sampling year to 2019. We used the eight-cell neighborhood rule, in which all eight adjacent pixels of the same class type are considered as members of the same feature, since the four-cell rule is conservative and may underestimate values (Turner & Gardner, 2015).
Minimum and average distances to fragment/fragments with the occurrence of allochthonous species (C. penicillata and/or C. jacchus) were calculated using the centroids (i.e., geometric center) of fragments ( Table 1). The variable altitude, on the contrary, was the average altitude of each fragment. Altimetric data were extracted from a TOPODATA digital elevation model (INPE, 2008) with 30-m resolution (Table 1). Both metrics were calculated on QGIS.
Two metrics, calculated on Fragstats, were used as a proxy to obtain data regarding connectivity: Euclidean Nearest Neighbor Distance (ENN) and the Proximity Index (PROX). Euclidean Nearest Neighbor Distance quantifies the Euclidean distance between the focal fragment and the fragments of the same class, based on the distance between the centroids of the two closest cells between fragments (McGarigal et al., 2015). The higher the ENN, the greater the isolation of a fragment compared with the others. The PROX metric (Gustafson & Parker, 1992) calculates, within a predefined search radius, the areas of fragments of the same class as the focal fragment, divided by proximity (i.e., edge-to-edge Euclidean distance from the focal fragment to the others). In this case, the search radius was defined as 1000 m, which corresponds to the average daily distance traveled by C. aurita (Corrêa et al., 2000). The higher the PROX value, the greater the presence of closer and more continuous fragments of the same class (McGarigal et al., 2015). An average was computed per sample fragment for both metrics, based on values from the sampling year to 2019 (Table 1).
Finally, for the surrounding matrix, a 5 km buffer was created around each forest fragment. The buffers were submitted to GRASS

| Data analysis
To assess whether landscape elements (i.e., patch attributes and landscape composition) and the presence of allochthonous species influence the probability of occurrence of C. aurita in forest fragments, we used generalized linear models (GLMs) with binomial distribution in the R environment version 4.0.5, using stats base package (R Core Team, 2021 the models to up to four variables (Doherty et al., 2012). This strategy resulted in a balanced set of models (i.e., all the variables were represented equally in the same number of models) that allowed the interpretation of the cumulative AICc weight (w + ) of each predictor variable (Burnham & Anderson, 2002). We considered variables to be determinant of the probability of occurrence of C. aurita to be those with w + ≥ 0.50 (Berger & Barbieri, 2004). We performed these analyses in R version 4.0.5, using the MuMIn package version 1.43.17 (Barton, 2020; Appendix S2).
The variable with the greatest influence on the probability of occurrence of C. aurita was minimum distance to fragment with C. jacchus (w + = 0.94; Table 3), followed by three variables referring to open and/or anthropic matrix categories in the surrounding matrix: agriculture and pasture mosaic percentage (w + = 0.61; Table 3), nonvegetated percentage (w + = 0.59; Table 3) and savanna formation percentage (w + = 0.59; Table 3). Minimum distance to fragment with C. jacchus ( Figure 4) and nonvegetated percentage ( Figure 4) were negatively correlated with C. aurita occurrence, while agriculture and pasture mosaic percentage and savanna formation percentage were positively correlated with C. aurita occurrence (Figure 4).

| DISCUSS ION
Landscape composition attributes such as matrix composition and the presence of allochthonous species influence C. aurita occurrence within its distribution area in Southeast Brazil. Among the 13 variables considered (Appendix S3), only minimal distance to forest fragments with C. jacchus and matrix categories of agriculture and pasture mosaic, nonvegetated, and savanna formation influenced the probability of C. aurita occurrence.

F I G U R E 3
Map of records for Callithrix aurita (black ball), C. penicillata (lozenge), C. jacchus (triangle), and control fragments (without the confirmed presence of C. aurita white ball), located within the study area.
Allochthonous species, especially C. jacchus, occur near and in hybrid zones together with C. aurita. We believe that the presence of C. jacchus does not favor the occurrence of C. aurita. In fact, the same environmental variables favor the occurrence of both species, which is critical considering the potential for hybridization and genetic introgression between the two (Carvalho et al., 2018;Malukiewicz et al., 2021). Expansions of the distributions of allochthonous species restrict areas of occurrence of C. aurita, making it difficult to conserve its genetic integrity (Carvalho et al., 2018).
The Callithrix genus speciation is relatively recent, which enables hybridization and some natural hybrid zones (Malukiewicz, 2019).
Nevertheless, some species would never hybridize naturally because of the distance and barriers (e.g., C. jacchus and C. aurita; Figure 1). Note: w + represents the sum of AICc weights of all models that contain the variable of interest. Variables with w + ≥ 0.50 were considered determinants (Berger & Barbieri, 2004). Estimates of variables (β parameters) were given by the most parsimonious model that included each variable. CI represents the 95% confidence interval. The β parameters are in logit scale.

TA B L E 3
Cumulative weight value of AICc (w + ) for each predictor variable evaluated as a possible influencer of the probability of Callithrix aurita occurrence.
groups of C. aurita turned into mixed groups and had hybrid offspring 5 years after C. jacchus and C. penicillata were introduced in Serra dos Órgãos National Park, state of Rio de Janeiro (Carvalho et al., 2018).
Considering that the data of the present study were generated on a time scale of almost 30 years, it is expected that currently only hybrids can be found in some areas. Although pure and isolated populations of C. aurita still exist, the advancement of allochthonous species and increased habitat fragmentation and conversion continue to press the species towards extinction (Carvalho et al., 2018;Vital et al., 2020).
Agriculture and pasture mosaic in the surrounding matrix positively influenced the probability of C. aurita occurrence. In these regions (Minas Gerais, São Paulo, and Rio de Janeiro), native fragments are largely surrounded by anthropic matrices, and this is part of the history of the Atlantic Forest deforestation (Fonseca, 1985), the Cerrado. Therefore, there would be areas of Cerrado phytophysiognomies interspersed with Atlantic Forest, such as gallery forests, which could favor the occurrence of the species. On the contrary, and as expected, nonvegetated areas in the surrounding matrix had a negative influence on the probability of C. aurita occurrence. As these areas do not have canopy formation, they do not provide refuges or feeding areas for arboreal species, nor even favor their movement (Arroyo-Rodríguez & Mandujano, 2009).
The other variables did not influence the probability of C. aurita occurrence. There was just a single record of the species using planted forest in the surrounding matrix (Norris et al., 2011), suggesting that that C. aurita may be more selective in the use of the habitat and typically avoids it. On the contrary, both the pasture matrix and the agriculture matrix present less structural heterogeneity when compared to the agriculture and pasture mosaic matrix, and the absence of its influence over C. aurita occurrence is corroborated by the study of Silva et al. (2015). There are still large, vegetated areas of forest formation in the landscape, which characterizes it as a non-limiting variable.
Furthermore, these areas can act as stepping-stones for the native species (Driscoll et al., 2013), but they can also serve as a gateway for allochthonous species (Alharbi & Petrovskii, 2018). Thus, it is essential that the factors that influence the occurrence of allochthonous species be evaluated to help mitigate their immigration.
The lack of an influence of fragment size corroborates Oliveira (2012), who recorded the occurrence of C. aurita in fragments of different magnitude of size. Furthermore, considering the relationship between home range and resource availability (Oliveira, 2012), landscape supplementation may be occurring in smaller fragments (Pozo-Montuy et al., 2013;Valença-Silva et al., 2014). On the contrary, the ENN metric, despite being very usual, ignores landscape components, which may represent a simplification of the system and, therefore, omit landscape attributes that may be more associated with landscape connectivity for C. aurita (Hargis et al., 1998). Altitude, despite being relevant on a refined scale (Norris et al., 2011), does not seem to have the same influence on a landscape scale. Callithrix aurita can be found over a large altitudinal range, even though most low-altitude areas have been decimated by human activities in recent centuries (Brandão & Develey, 1998 where only one group with C. aurita individuals were found, and all of the other groups we composed by hybrids (Guimarães-Lopes et al., Unpublished data). This means that we are losing the pure genetics of an already threatened species because of the biological invasions.
We hope that the results of this work can also contribute substantially to the conservation of other primate species that face the same challenges as C. aurita and are endangered, as is the case of C. flaviceps. This is possible once they can support the definition of priority areas to implement conservation plans for those primates, such as habitat restoration, and implementation of corridors between patches, also showing the necessity of new population surveys and censuses in some areas. All of these are in order to understand where we need to put the effort in rescuing the last pure individuals to ex situ management, and also do something about the biological invasions, with the purpose of preventing new colonization and the genetic mix between native and invasive species.

ACK N OWLED G M ENTS
We would like to thank Fabiano Melo and Leandro Jerusalinsky for their major contributions to this study. The main author also thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES for the scholarship received during this study. The authors are also grateful for the reviewers of the manuscript.

FU N D I N G I N FO R M ATI O N
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -CAPES (Grant 88887.342104/2019-00).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Dryad at https://doi.org/10.5061/dryad.8sf7m 0ctc.